Proc. of the 6th Animal Wealth Research Conf. in the Middle East & North Africa
EFFECT OF PROBIOTICS BACTERIA ISOLATED FROM
YOGHURTS PRODUCED IN DAMIETTA CITY ON SOME
PATHOGENIC BACTERIA
Hassan Ali Maarof; Abdallah, M.I.M; Bazalou, M.S. and
Abo-Samra, R.G.
Food Inspection Lab., Damietta Sea Port, Animal Health Research Institute,
ARC, Giza, Egypt
ABSTRACT
A
total of 200 samples of different brand of traditional (balady) yoghurts were
randomly collected from local markets and supermarkets in various districts of
Damietta city.
Probiotics were isolated from yoghurts samples anaerobically in the presence of
10% CO2 and identified. In this study a total of 17 lactic acid bacteria were isolated.
For the antimicrobial activity of the isolated probiotics organisms were used as the
inhibitory substances against some common foodborne pathogens (E.coli, Salmonella
spp. and Staphylococcus aureus) using the agar-well diffusion assay.
In conclusion, yoghurts probiotics has an antimicrobial activity in vitro against
the tested indicator pathogens and have better antimicrobial activity at concentration
of 109 CFU/g .
Keywords: probiotics, traditional (balady) yoghurts, antibacterial, E. coli,
Staphylococcus aureus and Salmonella spp.
Hurghada information center- 27-30 September (2013) - pp. 01 - 14
Effect of Probiotics bacteria isolated from yoghurts produced in Damietta city on ………
INTRODUCTION
Probiotics are widely used in fermented food production and are considered as
generally recognized as safe organisms which are safely applied in medical and
veterinary functions. In the food industry, probiotics are widely employed as starter
cultures and have been indexed as part of human microbiota. Yoghurts, cheese and
fermented milk products are mentioned as the main food sources of probiotics. The
lactic acid that is produced from the fermentation of lactose contributes to the sour
taste of yoghurts by decreasing pH and allows for the characteristic texture by acting
on the milk proteins Heller, (2001). Probiotics are alive and nonpathogenic
microorganisms that have beneficial effects on their host’s health. Traditional dairy
products have been used for many centuries among the natives and are the main
source of potentially probiotics bacteria.
Probiotics are living, health-promoting microorganisms that are incorporated
into various kinds of foods. The ability of probiotics to withstand the normal acidic
conditions of the gastric juices and the bactericidal properties of the bile salts, as well
as the production of lactic acid that inhibits the growth of other microorganisms,
allow them to be established in the intestinal tract (Oskar et al., 2004). Probiotics can
produce a wide range of antimicrobial metabolites, i.e. organic acids, diacetyl,
acetoin, hydrogen peroxide and bacteriocins. These antimicrobial activities can
contribute in the microbiological safety by controlling the growth of other
microorganisms, and inhibition of pathogenic bacteria Hobbs, (2000) and Ouweh and
Vest (2004). The reported health benefits of probiotics include: boosting of the
immune system, inhibition of the growth of pathogenic microorganisms, prevention
of diarrhea from various causes, prevention of cancer, reduction of the risk of
inflammatory bowel movements, improvement of digestion of proteins and fats,
synthesis of vitamins, and detoxification and protection from toxins Sonomoto and
Yokota (2011). Members of the genera Lactobacillus and Streptococcus are the most
common probiotics used in commercial fermented and non-fermented dairy products
today (Reid and Bruce 2006).
The aim of this study was to determine the antibacterial effects among the
probiotics bacteria isolated from different yoghurts marketed in Damietta Governorate
against some common bacterial food pathogens.
MATERIALS AND METHODS
Materials:
Samples collection:
Two hundred traditional (balady) yoghurts samples of (50 ml) were randomly
collected from different shops and super markets at Damietta city and kept under
refrigeration temperature using an ice box and brought to the laboratory.
39
Hassan Ali Maarof et al.
Methods:
Plating and isolation of lactic acid bacteria (LAB) according to the standards
IDF, International Standards (1996):
The samples of the yoghurts were shaken vigorously to suspend the bacterial
contents. Then, 10 g of each yoghurts sample was separately mixed in 50 ml of MRS
broth at pH 6.5. After mixing into MRS broth, they were shaken to obtain a
homogeneous suspension. Anaerobic condition in the presence of 10% CO2 was
created for removal of unwanted bacteria. The plates were incubated anaerobically on
jars using BBL, GasPak Plus at 37°C for 72 h. Finally, the single colony of bacteria
was isolated by observing their colonial morphology and some physiological tests
(Gram staining and catalase reaction).
Identification of isolates (Holt, 1984 and Ali 2011):
The suspected probiotics were further pure cultured for morphological and
biochemical identification. Gram stain and microscopic study were performed for the
isolates of 18 hrs. culture from MRS agar plates.
Acid tolerance test ( Laroia and Marin, 1991(:
The isolated Lactobacilli were subjected to primary screening for acid
tolerance in MRS broth adjusted to pH 2.5 with 1N HCl for 90 min at 37°C. The
determination of survival was performed by single streaking on MRS agar plates, and
the growth was observed after 24-48 h after anaerobic incubation at 37°C. Isolates
which were growing on the agar were considered to be acid tolerant strains.
Pathogenic bacterial strains:
The pathogenic strains used in this work were obtained from microbiology
laboratory, Animal Health Research Institute Giza, Cairo, Egypt. The pathogens were
maintained in brain heart infusion agar (BHIA) butt-slants in screw-capped tubes kept
at 4C. They included both Gram-negative (Escherichia coli, Salmonella spp.) and
Gram-positive strains (Staphylococcus aureus) (Laboratory strains).
Agar-well diffusion assay according to (Cardirici and Citak (2005); RaczyńskaCabaj et al., (2005) and Lalitha, (2007):
 The pathogenic bacterial strains from the stock cultures were subculture
aerobically in brain heart infusion broth (BHIB) medium at 37C for 18hrs.,
mixed at 1% with about 25ml of BHIA medium held at 45ºC (to give
approximately 106 CFU/ml), and then poured into sterile Petri dishes and
stored at 4ºC for 1h. to solidify the media.
 A 6mm wide well was cut in the center of each solidified agar plate, using a
sterile metal borer.
 Culture supernatants of probiotics isolates were prepared by cultivation
anaerobically in MRS broth overnight at 37°C and removal of the cells by
centrifugation at 2000×g for 10 minutes. Culture supernatants pH was adjusted
40
Effect of Probiotics bacteria isolated from yoghurts produced in Damietta city on ………








to 7.0 using (1M NaOH). Culture supernatant was passed through a sterile
microbiological filter (Millipore 0.22 μm) for sterilization, and then aliquots of
100 µl were poured in each well of BHIA medium.
The plates were first incubated at 4ºC for 1-2h. to allow the test material to
diffuse into the agar and, then incubated under aerobic conditions at 30ºC for
18-24hrs.
The previously prepared neutralized filtrate of cell-free supernatants were 2fold serially diluted in MRS broth and examined for their antimicrobial activity
as described before in order to determine the minimum inhibition
concentration (MIC) induced by each probiotics strain. MIC was expressed as
arbitrary unit (AU/ml), (one arbitrary unit was defined as the reciprocal of the
highest dilution showing a clear zone of growth inhibition on the indicator
lawn).
After 24h. of anaerobic incubation, each plate was examined for zones of
inhibition around the wells. Inhibition was considered positive when the width
of the clear zone around the was 0.5mm or more where, resistance was defined
as the absence of a growth inhibition zone around the well.
Finally, the diameter of the zones of complete inhibition was measured,
including the diameter of the disc. Zones are measured to the nearest whole
number in millimeter, using transparent ruler and recorded.
To further determine whether the selected pathogens were inhibited or killed
by probiotics, the growth inhibition zone was swabbed. The swab was then
inoculated into BHIB medium and incubated aerobically under 37°C for 24 h.
The BHIB medium were then checked for growth. The presence of growth in
the broth was interpreted as an inhibitory property in the agar plate, while no
growth was interpreted to be as a result of the bactericidal effect.
Each of the tests in the determination of antibacterial effects of the probiotics
was conducted in two trials, and in duplicate (Lim and Dond-Soon, 2009).
Parallel work was done as control without using the pathogenic bacteria.
Each of the tests in the determination of antimicrobial activity of the probiotics
was conducted in two trials, each in triplicate.
Minimal inhibitory concentration (MIC) and Minimal bactericidal concentration
(MBC) determination by broth dilution according to Abdelbasset and Djamila
(2008):
For MIC and MBC determinations, serial dilutions of culture supernatants of
probiotics in 5 ml of broth inoculated with 50 µl of fresh indicator pathogens
(inoculum, ∼108 CFU/ml). The tubes were incubated at 37°C overnight with shaking
and the highest dilution in which there were no growths was recorded as the MIC. For
MBC testing, aliquots (20 µl) of broth from tubes containing no growth were plated
onto solid medium and again incubated overnight at 37°C. The highest dilution in
which there were no survivors was recorded as the MBC. In the above method,
controls for each organism were performed using the sterile liquid medium without
aqueous crude extract. All MICs and MBCs were confirmed by triplicate assays.
41
Hassan Ali Maarof et al.
Determination of optimal growth and pH according to Kim et al., (2003):
For the determination of optimal growth and pH of Lactobacillus 1% (v/v)
fresh overnight culture of Lactobacillus were inoculated into MRS broth with varying
pH ranging from 2.5-8.5. The pH were adjusted with concentrated acetic acid (99%)
and 5N NaOH. The inoculated broths were incubated in anaerobic condition 24 h. at
37°C in the presence of 10% CO2. After 24 h of incubation growth of the bacteria
were measured using a spectrophometer, reading the optical density at 560 nm (OD)
against the uninoculated broth.
Statistical analysis:
Statistical analysis was performed using SPSS statistical program for windows
(Version 16) (SPSS Inc., Chicago, IL, and USA).
RESULTS AND DISCUSSION
After morphological examination in plates and Gram staining it could be
concluded that the isolated bacteria were rod shaped, convex, smooth, shiny,
irregular, circular, Gram positive, facultative anaerobic, non-spore forming which
indicate them to be the member of Lactobacillus spp (Holt, 1994). These isolates
were subjected to grow on selective MRS agar media and produced round shape, offwhite to cream color, shiny colonies those were quite similar to the reference
Lactobacillus spp. grown on MRS agar media. Isolates when Gram stained, found rod
shaped, short-medium chain and positive in Gram reaction those all are typical
characteristics of Lactobacillus spp. according to Bergey’s Manual of Determinative
Bacteriology (Holt, 1984).
Table (1): The number of rod-shaped (Lactobacillus isolates) after
biochemical identification and screening for acid
tolerance.
Dairy
Products
Samples
number
Number of isolates
Group 1
Group 2
Group 3
Group 4
50
50
50
50
200
2
5
3
7
17
42
Effect of Probiotics bacteria isolated from yoghurts produced in Damietta city on ………
Table (2): Diameter of zone of inhibition (mm) produced by
sterilized crude extract of probiotics isolates on
pathogenic bacteria using well diffusion assay
technique.
Group 1 isolates
(1)
(2)
-
-
-
-
-
E. COLI
SALMONELLA
S. AUREUS
12
20
9
9
12
10
-
-
-
-
-
Group 2 isolates
(1)
(2)
(3)
(4)
(5)
-
-
E. COLI
SALMONELLA
S. AUREUS
10
27
9
13
16
12
11
14
11
10
20
10
13
16
11
-
-
Group 3 isolates
(1)
(2)
(3)
-
-
-
-
E. COLI
SALMONELLA
S. AUREUS
13
17
9
12
14
10
13
14
9
-
-
-
-
Group 4 isolates
(1)
13
18
10
(2)
13
14
9
(3)
12
14
10
(4)
11
17
8
(5)
12
13
9
(6)
9
17
11
(7)
11
14
9
E. COLI
SALMONELLA
S. AUREUS
**Values between brackets denoted the probiotics isolates /examined groups
43
Hassan Ali Maarof et al.
Table (3): Antibacterial activity of probiotics isolates against three
pathogenic bacteria.
Antibacterial
Effect
MIC MBC
Group
Group 1
E. COLI
107
+ Ve
SALMONELLA
106
+ Ve
S. AUREUS
109
- Ve
E. COLI
107
+ Ve
SALMONELLA
106
+ Ve
S. AUREUS
109
- Ve
E. COLI
107
+ Ve
SALMONELLA
106
+ Ve
S. AUREUS
109
- Ve
E. COLI
107
+ Ve
SALMONELLA
106
+ Ve
S. AUREUS
109
- Ve
Group 2
Group 3
Group 4
44
Effect of Probiotics bacteria isolated from yoghurts produced in Damietta city on ………
The lactic acid bacteria (LAB) comprise a clade of Gram-positive, acidtolerant, generally non-sporulating, non-respiring rod or cocci that are associated by
their common metabolic and physiological characteristics. These bacteria, usually
found in decomposing plants and lactic products, produce lactic acid as the major
metabolic end-product of carbohydrate fermentation. Furthermore, lactic acid and
other metabolic products contribute to the organoleptic and textural profile of a food
item. The industrial importance of the LAB is further evinced by their generally
recognized as safe status, due to their ubiquitous appearance in food and their
contribution to the healthy microflora of human mucosal surfaces. Antimicrobial
activity is one of the most important selection criteria for probiotics. Antimicrobial
effects of lactic acid bacteria are incurred by producing some substances such as
organic acids (lactic, acetic, propionic acids), carbon dioxide, hydrogen peroxide,
diacetyl, low molecular weight antimicrobial substances and bacteriocins (Dunne et
al., 2001 and Ammor et al., 2006).
Data in table (1), showed the number of probiotics isolates which were 2, 5,
3 and 7 in group 1, 2, 3 and 4 respectively where all the isolates chosen were of the
Lactobacillus species without further confirmation by advanced methods such as
polymerase chain reaction.
The results showed in table (2), revealed that Lactobacilli probiotics
inhibited all of the test indicator microorganisms either Gram positive (S. aureus) or
Gram negative (E. coli and Salmonella) indicator pathogens and this data were in
coincide with that reported by Lei and Jacobsen, (2004) and Olivares et al., (2006).
From table (3), the results showed that isolated probiotics inhibited all of the
test indicator microorganisms; Lactobacilli spp were bactericidal for E. coli and
Salmonella spp., but inhibitory for S. aureus, the diameters of inhibition zones and it
could be concluded that the probiotics could inhibit the growth of indicator
microorganisms in different degrees as it had the strongest effect on Salmonella
followed by E. coli then at last was the effect on S. aureus where the widest inhibition
zone (27mm) was observed in case of Salmonella organisms subject to the inhibitory
effect of the first probiotics isolated from yoghurts sample in group 2 and in the same
time the narrowest inhibition zone was observed in case S. aureus subjected to the
inhibitory effect of probiotics isolate from yoghurts samples in group 4. These data
were nearly as previously obtained by Ayad et al., ( 2004) and Lei and Jacobsen,
(2004) in the study on the antimicrobial activity of the probiotics isolated from
yoghurts. Many authors supposed the antimicrobial effect of probiotics may be due to
the production of acetic and lactic acids that lowered the pH of the medium
Bezkorovainy (2002) and Mobarez et al., (2008). The probiotics bacteria may also
have competed for nutrients, Marteau (1990), simultaneously produced hydrogen
peroxide and bacteriocins that acted as antibiotic agents. Other than bacteriocins,
some are also capable of reuterine production that is known to act as an antibacterial
compound Ray (2000) and Meyer et al., (2007).
The spectrum of antimicrobial activity for the species suggested that the
inhibitory components were different (Raja et al., 2009). Similarly (Cadirici and
Citak, 2005; Girum et al., 2005 and Hami, (2011) observed varying degree of
inhibition of various foodborne pathogens by the culture filtrate of lactic acid bacteria,
45
Hassan Ali Maarof et al.
although these inhibitory substances produced by the lactic acid bacteria strains acts
differently on the pathogenic reference indicator strains, inhibitive substances
produced by the lactic acid bacteria can be generally protein (Vandenberg 1993 and
Moghaddam et al., 2006). Inhibition caused by hydrogen peroxide and organic acids
was ruled out as the producer strains were cultured anaerobically and the culture
supernatant was neutralized before assaying the antimicrobial activity. However, the
importance of the inhibition effect varies according to serotypes (Savadogo et al
2004 and Dhanasekaran et al., 2010).
Kurmann and Rasic (1991) suggested to achieve optimal potential
therapeutic effects, the number of probiotics organisms in a probiotics product should
meet a suggested minimum of >106 CFU/ml-1 while other authors agreed that >107
(
Davis et al., 1999 and Raczynska-Cabaj et al., 2005) could do the same effect mean
while108 CFU/ml-1 was satisfactory levels by (Kailasapathya and Rybka 1997).
Probiotics concentration of 109 CFU/g in our study was the more effective
probiotics concentration. Also, most of the probiotics lactobacilli in human foods are
supplied in highly concentrated forms containing more than 10 9 CFU/g. These finding
was in line with the observations of others who worked on fermented milk, Almaz et
al (1999) in Ethiopia, Savadogo et al., (2004) in fermented milk of Burkina Faso,
Ayad et al., (2004) in Egypt.
In this work the sterilized crude extracts of the isolated probiotics organisms
were used, therefore its action on pathogenic bacteria could be attributed to all of the
previously mentioned substances.
Finally, the capability of the probiotics incorporated in bioyoghurts to inhibit
the growth, or even kill certain selected pathogens confirms the health benefits
derived from the consumption of these yoghurts as indicated from table (3), that
showed that all probiotics isolates had a bactericidal effect on E. coli and Salmonella
species but had a bacteriostatic effect on S. aureus with a minimum inhibition
concentration of 106 ,107 , and 109 for E. coli, Salmonella and S. aureus successively.
Public health significance:
Probiotics are intended to assist the body’s naturally occurring gut microbiota.
Some probiotics preparations have been used to prevent diarrhea caused by
antibiotics, or as part of the treatment for antibiotic-related symbiosis Soomro et al.,
(2002). Studies have documented probiotics effects on a variety of gastrointestinal
and extraintestinal disorders, including inflammatory bowel disease (IBD), irritable
bowel syndrome (IBS), vaginal infections, and immune enhancement (Shah and
Dave, 2002). Some probiotics have also been investigated in relation to atopic
eczema, rheumatoid arthritis, and liver cirrhosis (Ong et al., 2006). Although there is
some clinical evidence for the role of probiotics in lowering cholesterol, the results
are conflicting. In general, the strongest clinical evidence for probiotics is related to
their use in improving gut health and stimulating immune function Tharmarja and
Shah (2004). A number of studies have found probiotics consumption to be useful in
the treatment of many types of diarrhea, including antibiotic-associated diarrhea in
adults, travelers' diarrhea, and diarrheal diseases in young children caused by
rotaviruses. Lactobacillus and Streptococcus are the most common probiotics used in
46
Effect of Probiotics bacteria isolated from yoghurts produced in Damietta city on ………
commercial fermented and non-fermented dairy products today (Heller, 2001).
Antimicrobial effects of lactic acid bacteria are formed by producing some substances
such as organic acids (lactic, acetic, propionic acids), carbon dioxide, hydrogen
peroxide, diacetyl, low molecular weight antimicrobial substances and bacteriocins
(Quwehand and Vesterlund, 2004).
Lactic acid bacteria (LAB) have been used successfully, with few adverse
effects, to prevent antibiotic associated diarrhea, to treat acute infantile diarrhea and
recurrent Clostridium disease and to treat various diarrheal illnesses, the antagonistic
property is attributed to the lowered pH, the undissociated acids and production of
other primary and secondary antimicrobial metabolites produced by LAB. The
metabolites produced by the fermentation process, except the volatile ones, are kept in
the foods and result in growth inhibition of food spoilage or poisoning bacteria and
detoxification of noxious compounds of plant origin (Reidand and Bruce 2006). The
primary antimicrobial effect exerted by LAB is the production of lactic acid and
reduction of pH. In addition, LAB produce various antimicrobial compounds, which
can be classified as low-molecular-mass (LMM) compounds such as hydrogen
peroxide (H2O2), carbon dioxide (CO2), diacetyl (2,3-butanedione), uncharacterized
compounds, and high-molecular-mass (HMM) compounds like bacteriocin (9-12). All
of these can antagonize the growth of some spoilage and pathogenic bacteria in foods
(Catanzaro and Green, (1997); Moghaddam et al., (2006) and Aweenet et al.,
(2012).
In general the reported health benefits include: boosting of the immune system,
inhibition of the growth of pathogenic microorganisms, prevention of diarrhea from
various causes, prevention of cancer, reduction of the risk of inflammatory bowel
movements, improvement of digestion of proteins and fats, synthesis of vitamins, and
detoxification and protection from toxins (Hobbs, 2000).
CONCLUSION
The probiotic bacteria isolated in our study possess varying degrees of
inhibition towards tested enteric pathogenic bacteria to indicate that there is potential
for the derivation of health benefits from consuming traditional yoghurts containing
specific strains of probiotics are safe for human use and able to confer some health
benefits on the host. The health benefits for which probiotics can be applied include
conditions such as gastrointestinal infections, certain bowel disorders, allergy, and
urogenital infections, which afflict a large portion of the world’s population.
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of lactic acid bacteria
Sonomoto, K and Yokota, A. (editor) (2011): Lactic acid Bacteria: Antimicrobial
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Tharmaraj, N. and Shah, N. P.(2004): Antimicrobial effects of probiotics against
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interference with microbial growth. FEMS Microbiology Reviews 12: 221238.
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‫……… ‪Effect of Probiotics bacteria isolated from yoghurts produced in Damietta city on‬‬
‫"تأثير بكتيريا البروبيوتك المعزولة من الزبادي المنتج بمحافظة دمياط على بعض‬
‫الميكروبات الممرضة"‬
‫د‪ /‬حسن علي معروف‪ -‬د‪/‬مدحت إبراهيم عبدهللا‪-‬د‪/‬محمد صالح البدري بزالو‪-‬د‪ /‬راندا جبر أبو سمره‬
‫معمل فحوص األغذية بميناء دمياط البحري‪-‬معهد بحوث صحه الحيوان–مركز البحوث الزراعية‬
‫الملخص‬
‫جمعت ما مجموعه ‪ 022‬عينة من مختلف العالمات التجارية من الزبادي التقليدية (بلدي)‬
‫بشكل عشوائي من األسواق المحلية ومحالت السوبر ماركت في أحياء مختلفة من مدينة‬
‫دمياط‪.‬‬
‫تممم عممز ممما مجموعممه ‪ 71‬مممن معممزو ت بكتيريمما البروبيوتيممم مممن عينممات الزبممادي التقليديممة‬
‫هوائيمما فممي وجممود ‪ ٪72‬ثمماني اكسمميد الكربممون تممم الكشممف عممن النشمماط البكتيممري لبكتيريمما‬
‫البروبيوتيم المعزولة عمن الممواد المطب مة ضمد بعمم مسمببات األممرات التمي تنقلهما األغذيمة‬
‫الش مائعة مطممل السممالمونيال واوي كممو ي والميكممرو العنقممودي الممذهبي باسممتخداا ابتبممار نشممر‬
‫اآلجار وقد أظهرت هذه ا بتبارات عمن التمثثير التطبي مي الجيمد لمعمزو ت البروبيوتيمم ضمد‬
‫المكروبات المرضية محل البحث بشكل جيد أظهرت النتائج أن بكتيريا البروبيوتيم الموجودة‬
‫في عينات الزبمادي المختبمرة لهما تمثثير مطمب جيمد فمي المختبمر ضمد بعمم مسمببات األممرات‬
‫الممرضة المختبرة ويكون أفضل تثثير لها عند تركيز ‪/972‬جم‪.‬‬
‫كلمااات البحا الدالااة‪ :‬بكتيريمما البروبيوتيممم التقليديممة الزبممادي البلممدض مضمماد للجممراثيم اوي‬
‫كو ي القولونية المكورات العنقودية الذهبية السالمونيال محافظه دمياط‪.‬‬
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